Treatment of cardiovascular and related pathologies

ABSTRACT

Methods for treating cardiovascular and related diseases such as ischemia, ischemia reperfusion injuries, and myocardial ischemia, are described. The methods are directed to concurrently administering a compound such as pyridoxal-5′-phosphate, pyridoxamine, pyridoxal, or a 3-acylated pyridoxal analogue with a therapeutic cardiovascular compound.

[0001] This application claims priority to provisional applicationSerial No. 60/150,415 filed on Aug. 24, 1999. This application is adivisional of application Ser. No. 09/645,237 filed on Aug. 24, 2000.

FIELD OF THE INVENTION

[0002] This invention relates to methods of treating cardiovascular andrelated diseases, such as hypertrophy, hypertension, congestive heartfailure, ischemia, such as myocardial ischemia, ischemia reperfusioninjuries in various organs, arrhythmia, and myocardial infarction.

BACKGROUND

[0003] Heart failure is a pathophysiological condition in which theheart is unable to pump blood at a rate commensurate with therequirement of the metabolizing tissues or can do so only from anelevated filling pressure (increased load). Thus, the heart has adiminished ability to keep up with its workload. Over time, thiscondition leads to excess fluid accumulation, such as peripheral edema,and is referred to as congestive heart failure.

[0004] When an excessive pressure or volume load is imposed on aventricle, myocardial hypertrophy (i.e., enlargement of the heartmuscle) develops as a compensatory mechanism. Hypertrophy permits theventricle to sustain an increased load because the heart muscle cancontract with greater force. However, a ventricle subjected to anabnormally elevated load for a prolonged period eventually fails tosustain an increased load despite the presence of ventricularhypertrophy, and pump failure may ultimately occur.

[0005] Heart failure can arise from any disease that affects the heartand interferes with circulation. For example, a disease that increasesthe heart muscle's workload, such as hypertension, will eventuallyweaken the force of the heart's contraction. Hypertension is a conditionin which there is an increase in resistance to blood flow through thevascular system. This resistance leads to increases in systolic and/ordiastolic blood pressures. Hypertension places increased tension to theleft ventricular myocardium, causing it to stiffen and hypertrophy, andaccelerates the development of atherosclerosis in the coronary arteries.The combination of increased demand and lessened supply increases thelikelihood of myocardial ischemia leading to myocardial infarction,sudden death, arrhythmias, and congestive heart failure.

[0006] Ischemia is a condition in which an organ or a part of the bodyfails to receive a sufficient blood supply. When an organ is deprived ofits blood supply, it is said to be hypoxic. An organ will become hypoxiceven when the blood supply temporarily ceases, such as during a surgicalprocedure or during temporary artery blockage. Ischemia initially leadsto a decrease in or loss of contractile activity. When the organaffected is the heart, this condition is known as myocardial ischemia,and myocardial ischemia initially leads to abnormal electrical activity.This may generate an arrhythmia. When myocardial ischemia is ofsufficient severity and duration, cell injury may progress to celldeath-i.e., myocardial infarction-and subsequently to heart failure,hypertrophy, or congestive heart failure.

[0007] When blood flow resumes to an organ after temporary cessation,this is known as ischemic reperfusion of the organ. For example,reperfusion of an ischemic myocardium may counter the effects ofcoronary occlusion, a condition that leads to myocardial ischemia.Ischemic reperfusion to the myocardium may lead to reperfusionarrhythmia or reperfusion injury. The severity of reperfusion injury isaffected by numerous factors, such as, for example, duration ofischemia, severity of ischemia, and speed of reperfusion. Conditionsobserved with ischemia reperfusion injury include neutrophilinfiltration, necrosis, and apoptosis.

[0008] Drug therapies, using known active ingredients such asvasodilators, angiotensin II receptor antagonists, angiotensinconverting enzyme inhibitors, diuretics, antithrombolytic agents,β-adrenergic receptor antagonists, α-adrenergic receptor antagonists,calcium channel blockers, and the like, are available for treating heartfailure and associated diseases. Of course, any drug used for treatmentmay result in side effects. For example, vasodilators may result inhypotension, myocardial infarction, and adverse immune response.Angiotensin II receptor antagonists and angiotensin converting enzymeinhibitors are often associated with acute renal failure, fetopathicpotential, proteinuria, hepatotoxicity, and glycosuria as side effects.Similarly, common side effects associated with calcium channel blockersinclude hypotension, peripheral edema, and pulmonary edema. β-Adrenergicreceptor antagonists and diuretics have been associated withincompatibility with nonsteroidal anti-inflammatory drugs in addition toimpotence, gout, and muscle cramps in the case of diuretics and inaddition to a decrease in left ventricular function and suddenwithdrawal syndrome in the case of β-adrenergic receptor antagonists.Moreover, side effects associated with α-adrenergic receptor antagonistsinclude thostatic hypotension, and side effects associated withantithrombolytic agents include excessive bleeding.

[0009] To address the side effects, the dosage of a drug may be reducedor the administration of the drug may be abated and replaced withanother drug. It would be desirable to administer a drug therapy withdecreased amounts of the active ingredient to reduce side effects butmaintain effectiveness.

SUMMARY OF THE INVENTION

[0010] The present invention provides methods for treatingcardiovascular and related diseases, such as, for example, hypertrophy,hypertension, congestive heart failure, myocardial ischemia, ischemiareperfusion injuries in an organ, arrhythmia, and myocardial infarction.One embodiment is directed to a method of treating cardiovasculardisease in a mammal by concurrently administering to the mammal atherapeutically effective amount of a combination of a compound suitablefor use in methods of the invention and a therapeutic cardiovascularcompound. Therapeutic cardiovascular compounds suitable for use inmethods of the invention include an angiotensin converting enzymeinhibitor, an angiotensin II receptor antagonist, a calcium channelblocker, an antithrombolytic agent, a β-adrenergic receptor antagonist,a vasodilator, a diuretic, an α-adrenergic receptor antagonist, anantioxidant, and a mixture thereof. In some embodiments, the therapeuticcardiovascular compound is PPADS.

[0011] Compounds suitable for use in the methods of the inventioninclude pyridoxal-5′-phosphate, pyridoxamine, pyridoxal, 3-acylatedpyridoxal analogues, pharmaceutically acceptable acid addition saltsthereof, and mixtures thereof.

[0012] In one embodiment, a 3-acylated pyridoxal analogue is a compoundof the formula

[0013] In another embodiment, a 3-acylated pyridoxal analogue is acompound of the formula

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1 is a graph showing the effect of P-5-P and aspirin, aloneor in combination, on mortality in the rat model of coronary ligation.

[0015]FIG. 2 is a graph showing the effect of P-5-P and captopril, aloneor in combination, on mortality in the rat model of coronary ligation.

[0016]FIG. 3 is a graph showing the effect of P-5-P and propranolol,alone or in combination, on mortality in the rat model of coronaryligation.

[0017]FIG. 4 is a graph showing the effect of P-5-P and verapamil, aloneor in combination, on mortality in the rat model of coronary ligation.

[0018]FIG. 5 is a graph showing the effect of P-5-P and aspirin, aloneor in combination, on scar weight in the rat model of coronary ligation.

[0019]FIG. 6 is a graph showing the effect of P-5-P and captopril, aloneor in combination, on scar weight in the rat model of coronary ligation.

[0020]FIG. 7 is a graph showing the effect of P-5-P and propranolol,alone or in combination, on scar weight in the rat model of coronaryligation.

[0021]FIG. 8 is a graph showing the effect of P-5-P and verapamil, aloneor in combination, on scar weight in the rat model of coronary ligation.

[0022]FIG. 9 is a graph showing the effect of P-5-P and aspirin, aloneor in combination, on the rate of force of contraction (+dp/dt) in therat model of coronary ligation.

[0023]FIG. 10 is a graph showing the effect of P-5-P and captopril,alone or in combination, on the rate of force of contraction (+dp/dt) inthe rat model of coronary ligation.

[0024]FIG. 11 is a graph showing the effect of P-5-P and propranolol,alone or in combination, on the rate of force of contraction (+dp/dt) inthe rat model of coronary ligation.

[0025]FIG. 12 is a graph showing the effect of P-5-P verapamil, alone orin combination, on the rate of force of contraction (+dp/dt) in the ratmodel of coronary ligation.

[0026]FIG. 13 is a graph showing the effect of P-5-P and aspirin, aloneor in combination, on the rate of force of relaxation (−dp/dt) in therat model of coronary ligation.

[0027]FIG. 14 is a graph showing the effect of P-5-P and captopril,alone or in combination, on the rate of force of relaxation (−dp/dt) inthe rat model of coroary ligation.

[0028]FIG. 15 is a graph showing the effect of P-5-P and propranolol,alone or in combination, on the rate of force of relaxation (−dp/dt) inthe rat model of coronary ligation.

[0029]FIG. 16 is a graph showing the effect of P-5-P and verapamil,alone or in combination, on the rate of force of relaxation (−dp/dt) inthe rat model of coronary ligation.

[0030]FIG. 17 is a graph showing the effect of P-5-P and aspirin, aloneor in combination, on left ventricular end diastolic pressure (LVEDP) inthe rat model of coronary ligation.

[0031]FIG. 18 is a graph showing the effect of P-5-P and captopril,alone or in combination, on left ventricular end diastolic pressure(LVEDP) in the rat model of coronary ligation.

[0032]FIG. 19 is a graph showing the effect of P-5-P and propranolol,alone or in combination, on left ventricular end diastolic pressure(LVEDP) in the rat model of coronary ligation.

[0033]FIG. 20 is a graph showing the effect of P-5-P and verapamil,alone or in combination, on left ventricular end diastolic pressure(LVEDP) in the rat model of coronary ligation.

[0034]FIG. 21 is a graph showing the effect of P-5-P and aspirin, aloneor in combination, on heart weight in the rat model of coronaryligation.

[0035]FIG. 22 is a graph showing the effect of P-5-P and captopril,alone or in combination, on heart weight in the rat model of coronaryligation.

[0036]FIG. 23 is a graph showing the effect of P-5-P propranolol, aloneor in combination, on heart weight in the rat model of coronaryligation.

[0037]FIG. 24 is a graph showing the effect of P-5-P and verapamil,alone or in combination, on heart weight in the rat model of coronaryligation.

[0038]FIG. 25 is a graph showing the effect of P-5-P and aspirin, aloneor in combination, on right ventricular weight in the rat model ofcoronary ligation.

[0039]FIG. 26 is a graph showing the effect of P-5-P and captopril,alone or in combination, on right ventricular weight in the rat model ofcoronary ligation.

[0040]FIG. 27 is a graph showing the effect of P-5-P and propranolol,alone or in combination, on right ventricular weight in the rat model ofcoronary ligation.

[0041]FIG. 28 is a graph showing the effect of P-5-P and verapamil,alone or in combination, on right ventricular weight in the rat model ofcoronary ligation.

[0042]FIG. 29A is a graph showing systolic blood pressure in rats fromall pretreatment experiment groups at “0” day. “C” designates a controlgroup; “S” designates a sucrose diet induced diabetic group; “M”designates a group administered P-5-P alone; “Ca” designates a groupadministered captopril alone; “V” designates a group administeredverapamil alone; “M+Ca” designates a group administered P-5-P andcaptopril; “M+V” designates a group administered P-5-P and verapamil.

[0043]FIG. 29B is a graph showing the effect of pretreatment with P-5-P,captopril and verapamil on systolic blood pressure in rats whenadministered 1 week prior to sucrose diet induced diabetes. “C”, “S”,“M”, “Ca”, “V”, “M+Ca”, and “M+V” are designated as in FIG. 29A.

[0044]FIG. 30A is a graph showing systolic blood pressure in rats fromall experiment groups involved in same day treatment as sucrose feedingat “0” day. “C”, “S”, “M”, “Ca”, “V”, “M+Ca”, and “M+V” are designatedas in FIG. 29A.

[0045]FIG. 30B is a graph showing the effect of administration of P-5-P,captopril and verapamil on systolic blood pressure in rats whenadministered the same day as sucrose feeding to induce diabetes. “C”,“S”, “M”, “Ca”, “V”, “M+Ca”, and “M+V” are designated as in FIG. 29A.

[0046]FIG. 31A is a graph showing systolic blood pressure in rats fromall experiment groups involved in treatment two weeks after sucrosefeeding at “0” day. “C”, “S”, “M”, “Ca”, “V”, “M+Ca”, and “M+V” aredesignated as in FIG. 29A.

[0047]FIG. 31B is a showing systolic blood pressure in rats from allexperiment groups involved in treatment two weeks after sucrose feedingat “0” day. “C”, “S”, “M”, “Ca”, “V”, “M+Ca”, and “M+V” are designatedas in FIG. 29A.

DESCRIPTION OF THE INVENTION

[0048] The present invention provides methods for treatment ofcardiovascular and related diseases or conditions. Such cardiovascularand related diseases include hypertrophy, hypertension, congestive heartfailure, ischemia, such as myocardial ischemia, ischemia reperfusioninjury, arrhythmia, and myocardial infarction.

[0049] In accordance with the present invention, it has been found thatpyridoxal-5′-phosphate and its derivatives can be used concurrently withtherapeutic cardiovascular compounds in the treatment of theabove-identified diseases and conditions. “Treatment” and “treating” asused herein include preventing, inhibiting, and alleviatingcardiovascular diseases, related diseases, and related symptoms as wellas healing the ischemia-related conditions or symptoms thereof affectingmammalian organs and tissues. Treatment may be carried out byconcurrently administering a therapeutically effective amount of acombination of a compound suitable for use in methods of the inventionand a therapeutic cardiovascular compound.

[0050] A “therapeutically effective amount” as used herein includes aprophylactic amount, for example, an amount effective for preventing orprotecting against cardiovascular diseases, related diseases, andsymptoms thereof, and amounts effective for alleviating or healingcardiovascular diseases, related diseases, and symptoms thereof. Byadministering a compound suitable for use in methods of the inventionconcurrently with a therapeutic cardiovascular compound, the therapeuticcardiovascular compound may be administered in a dosage amount that isless than the dosage amount required when the therapeutic cardiovascularcompound is administered as a sole active ingredient. By administeringlower dosage amounts of the active ingredient, the side effectsassociated therewith should accordingly be reduced.

[0051] Compounds suitable for use in the methods of the inventioninclude pyridoxal-5′-phosphate, pyridoxal, pyridoxamine, 3-acylatedpyridoxal analogues, pharmaceutically acceptable acid addition saltsthereof, and mixtures thereof. 3-Acylated pyridoxal analogues providefor slower metabolism to pyridoxal in vivo. For example, a suitable3-acylated analogue of pyridoxal(2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine) is a compound ofthe formula I:

[0052] or a pharmaceutically acceptable acid addition salt thereof,wherein

[0053] R₁ is a straight or branched alkyl group, a straight or branchedalkenyl group, in which an alkyl or alkenyl group may be interrupted bya nitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or anunsubstituted or substituted aryl group.

[0054] The term “alkyl” group includes a straight or branched saturatedaliphatic hydrocarbon chain having from 1 to 8 carbon atoms, such as,for example, methyl, ethyl, propyl, isopropyl (1-methylethyl), butyl,tert-butyl (1,1-dimethylethyl), and the like.

[0055] The term “alkenyl” group includes an unsaturated aliphatichydrocarbon chain having from 2 to 8 carbon atoms, such as, for example,ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-methyl-1-propenyl, and thelike.

[0056] The above alkyl or alkenyl groups may optionally be interruptedin the chain by a heteroatom, such as, for example, a nitrogen or oxygenatom, forming an alkylaminoalkyl or alkoxyalkyl group, for example,methylaminoethyl or methoxymethyl, and the like.

[0057] The term “alkoxy” group includes an alkyl group as defined abovejoined to an oxygen atom having preferably from 1 to 4 carbon atoms in astraight or branched chain, such as, for example, methoxy, ethoxy,propoxy, isopropoxy (1-methylethoxy), butoxy, tert-butoxy(1,1-dimethylethoxy), and the like.

[0058] The term “dialkylamino” group includes two alkyl groups asdefined above joined to a nitrogen atom, in which the alkyl group haspreferably 1 to 4 carbon atoms, such as, for example, dimethylamino,diethylamino, methylethylamino, methylpropylamino, diethylamino, and thelike.

[0059] The term “aryl” group includes an aromatic hydrocarbon group,including fused aromatic rings, such as, for example, phenyl andnaphthyl. Such groups may be unsubstituted or substituted on thearomatic ring by, for example, an alkyl group of 1 to 4 carbon atoms, analkoxy group of 1 to 4 carbon atoms, an amino group, a hydroxy group, oran acetyloxy group.

[0060] Preferred R₁ groups for compounds of formula I are toluyl ornaphthyl. Such R₁ groups when joined with a carbonyl group form an acylgroup

[0061] which preferred for compounds of formula I include toluoyl orβ-naphthoyl. Of the toluoyl group, the p-isomer is more preferred.

[0062] Examples of 3-acylated analogues of pyridoxal include, but arenot limited to, 2-methyl-3-toluoyloxy-4-formyl-5-hydroxymethylpyridineand 2-methyl-βnaphthoyloxy-4-formyl-5-hydroxymethylpyridine.

[0063] Another suitable analogue is a 3-acylated analogue ofpyridoxal-4,5-aminal (1-secondaryamino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine) of the formulaII:

[0064] or a pharmaceutically acceptable acid addition salt thereof,wherein

[0065] R₁ is a straight or branched alkyl group, a straight or branchedalkenyl group, in which an alkyl or alkenyl group may be interrupted bya nitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or anunsubstituted or substituted aryl group; and

[0066] R₂ is a secondary amino group.

[0067] The terms “alkyl,” “alkenyl,” “alkoxy,” “dialkylamino,” and“aryl” are as defined above.

[0068] The term “secondary amino” group includes a group of the formulaIII:

[0069] derived from a secondary amine R₃R₄NH, in which R₃ and R₄ areeach independently alkyl, alkenyl, cycloalkyl, aryl, or, when R₃ and R₄are taken together, may form a ring with the nitrogen atom and which mayoptionally be interrupted by a heteroatom, such as, for example, anitrogen or oxygen atom. The terms “alkyl,” “alkenyl,” and “aryl” areused as defined above in forming secondary amino groups such as, forexample, dimethylamino, methylethylamino, diethylamino, dialkylamino,phenylmethylamino, diphenylamino, and the like.

[0070] The term “cycloalkyl” refers to a saturated hydrocarbon havingfrom 3 to 8 carbon atoms, preferably 3 to 6 carbon atoms, such as, forexample, cyclopropyl, cyclopentyl, cyclohexyl, and the like.

[0071] When R₃ and R₄ are taken together with the nitrogen atom, theymay form a cyclic secondary amino group, such as, for example,piperidino, and, when interrupted with a heteroatom, includes, forexample, piperazino and morpholino.

[0072] Preferred R₁ groups for compounds of formula II include toluyl,e.g., p-toluyl, naphthyl, tert-butyl, dimethylamino, acetylphenyl,hydroxyphenyl, or alkoxy, e.g., methoxy. Such R₁ groups when joined witha carbonyl group form an acyl group

[0073] which preferred for compounds of formula II include toluoyl,β-naphthoyl, pivaloyl, dimethylcarbamoyl, acetylsalicyloyl, salicyloyl,or alkoxycarbonyl. A preferred secondary amino group may be morpholino.

[0074] Examples of 3-acylated analogues of pyridoxal-4,5-aminal include,but are not limited to,1-morpholino-1,3-dihydro-7-(p-toluoyloxy)-6-methylfuro(3,4-c)pyridine;1-morpholino-1,3-dihydro-7-(β-naphthoyloxy)-6-methylfuro(3,4-c)pyridine;1-morpholino-1,3-dihydro-7-pivaloyloxy-6-methylfuro(3,4-c)pyridine;1-morpholino-1,3-dihydro-7-carbamoyloxy-6-methylfuro(3,4-c)pyridine; and1-morpholino-1,3-dihydro-7-acetylsalicyloxy-6-methylfuro(3,4-c)pyridine.

[0075] The compounds of formula I may be prepared by reacting pyridoxalhydrochloride with an acyl halide in an aprotic solvent. A suitable acylgroup is

[0076] wherein R₁ is as defined above. A particularly suitable acylhalide includes p-toluoyl chloride or β-naphthoyl chloride. A suitableaprotic solvent includes acetone, methylethylketone, and the like.

[0077] The compounds of formula II may be prepared by reacting1-secondary amino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine withan acyl halide in an aprotic solvent. An acyl group is

[0078] wherein R₁ is as defined above. A particularly suitable acylhalide includes p-toluoyl chloride, β-naphthoyl chloride,trimethylacetyl chloride, dimethylcarbamoyl chloride, andacetylsalicyloyl chloride. A particularly suitable secondary amino groupincludes morpholino.

[0079] The compound1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine may beprepared by methods known in the art, for example, by reactingmorpholine and pyridoxal hydrochloride at a temperature of about 100° C.in a solvent. A suitable solvent includes, for example, toluene.Similarly, other secondary amines as defined for R₂ may be used asreactants to prepare the appropriate 1-secondary amino compounds.

[0080] The compounds of formula I may alternatively be prepared from thecompounds of formula II by reacting a compound of formula II with anaqueous acid, such as, for example, aqueous acetic acid.

[0081] One skilled in the art would recognize variations in the sequenceand would recognize variations in the appropriate reaction conditionsfrom the analogous reactions shown or otherwise known that may beappropriately used in the above-described processes to make thecompounds of formulas I and II herein.

[0082] The products of the reactions described herein are isolated byconventional means such as extraction, distillation, chromatography, andthe like.

[0083] Pharmaceutically acceptable acid addition salts of compoundssuitable for use in methods of the invention include salts derived fromnontoxic inorganic acids such as hydrochloric, nitric, phosphoric,sulfuric, hydrobromic, hydriodic, hydrofluoric, phosphorous, and thelike, as well as the salts derived from nontoxic organic acids, such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids,aliphatic and aromatic sulfonic acids, etc. Such salts thus includesulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate,propionate, caprylate, isobutyrate, oxalate, malonate, succinate,suberate, sebacate, fumarate, maleate, mandelate, benzoate,chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Also contemplated aresalts of amino acids such as arginate and the like and gluconate,galacturonate, n-methyl glutamine, etc. (see, e.g., Berge et al., J.Pharmaceutical Science, 66: 1-19 (1977).

[0084] The acid addition salts of the basic compounds are prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce the salt in the conventional manner. The free base formmay be regenerated by contacting the salt form with a base and isolatingthe free base in the conventional manner. The free base forms differfrom their respective salt forms somewhat in certain physical propertiessuch as solubility in polar solvents, but otherwise the salts areequivalent to their respective free base for purposes of the presentinvention.

[0085] Methods of the invention include concurrently administeringpyridoxal-5′-phosphate, pyridoxamine, pyridoxal, a 3-acylated pyridoxalanalogue, a pharmaceutically acceptable acid addition salt thereof, or amixture thereof with a therapeutic cardiovascular compound to treathypertrophy, hypertension, congestive heart failure, ischemia, such asmyocardial ischemia, ischemia reperfusion injury, arrhythmia, ormyocardial infarction. Preferably, the cardiovascular disease treated ishypertrophy or congestive heart failure. Still preferably, thecardiovascular disease treated is arrhythmia. Also preferably, thecardiovascular disease treated is ischemia reperfusion injury.

[0086] Therapeutic cardiovascular compounds that may be concurrentlyadministered with a compound suitable for use in methods of theinvention include an angiotensin converting enzyme inhibitor, anangiotensin II receptor antagonist, a calcium channel blocker, anantithrombolytic agent, a β-adrenergic receptor antagonist, avasodilator, a diuretic, an α-adrenergic receptor antagonist, anantioxidant, and a mixture thereof. A compound suitable for use inmethods of the invention also may be concurrently administered withPPADS (pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid), also atherapeutic cardiovascular compound, or with PPADS and another knowntherapeutic cardiovascular compound as already described. In a preferredembodiment, pyridoxal-5′-phosphate is concurrently administered withPPADS or with PPADS and another known therapeutic cardiovascularcompound, preferably an angiotensin converting enzyme inhibitor or anangiotensin II receptor antagonist.

[0087] Preferably, a therapeutic cardiovascular compound, which isconcurrently administered with pyridoxal-5′-phosphate, pyridoxamine,pyridoxal, a 3-acylated pyridoxal analogue, a pharmaceuticallyacceptable acid addition salt thereof, or a mixture thereof, is anangiotensin converting enzyme inhibitor, an angiotensin II receptorantagonist, or a diuretic. Still preferably, the therapeuticcardiovascular compound is an α-adrenergic receptor antagonist. Alsopreferably, the therapeutic cardiovascular compound is a calcium channelblocker.

[0088] These therapeutic cardiovascular compounds are generally used totreat cardiovascular and related diseases as well as symptoms thereof. Askilled physician or veterinarian readily determines a subject who isexhibiting symptoms of any one or more of the diseases described aboveand makes the determination about which compound is generally suitablefor treating specific cardiovascular conditions and symptoms.

[0089] For example, myocardial ischemia may be treated by theadministration of, for example, angiotensin converting enzyme inhibitor,an angiotensin II receptor antagonist, a calcium channel blocker, anantithrombolytic agent, a β-adrenergic receptor antagonist, a diuretic,an α-adrenergic receptor antagonist, or a mixture thereof. In someinstances, congestive heart failure may be treated by the administrationof, for example, angiotensin converting enzyme inhibitor, an angiotensinII receptor antagonist, a calcium channel blocker, a vasodilator, adiuretic, or a mixture thereof.

[0090] Myocardial infarction may be treated by the administration of,for example, angiotensin converting enzyme inhibitor, a calcium channelblocker, an antithrombolytic agent, a β-adrenergic receptor antagonist,a diuretic, an α-adrenergic receptor antagonist, or a mixture thereof.

[0091] Hypertension may be treated by the administration of, forexample, angiotensin converting enzyme inhibitor, a calcium channelblocker, a β-adrenergic receptor antagonist, a vasodilator, a diuretic,an α-adrenergic receptor antagonist, or a mixture thereof.

[0092] Moreover, arrhythmia may be treated by the administration of, forexample, a calcium channel blocker, a β-adrenergic receptor antagonist,or a mixture thereof.

[0093] Antithrombolytic agents are used for reducing or removing bloodclots from arteries.

[0094] Hypertropy may be treated by the administration of, for example,an angiotensin converting enzyme inhibitor, an angiotensin II receptorantagonist, a calcium channel blocker, or a mixture thereof.

[0095] Ischemia reperfusion injury may be treated by the administrationof, for example, an angiotensin converting enzyme inhibitor, anangiotensin II receptor antagonist, a calcium channel blocker, or amixture thereof.

[0096] Known angiotensin converting enzyme inhibitors include, forexample, captopril, enalapril, lisinopril, benazapril, fosinopril,quinapril, ramipril, spirapril, imidapril, and moexipril.

[0097] Examples of known angiotensin II receptor antagonists includeboth angiotensin I receptor subtype antagonists and angiotensin IIreceptor subtype antagonists. Suitable angiotensin II receptorantagonists include losartan and valsartan.

[0098] Suitable calcium channel blockers include, for example,verapamil, diltiazem, nicardipine, nifedipine, amlodipine, felodipine,nimodipine, and bepridil.

[0099] Antithrombolytic agents known in the art include antiplateletagents, aspirin, and heparin.

[0100] Examples of known β-adrenergic receptor antagonists includeatenolol, propranolol, timolol, and metoprolol.

[0101] Suitable vasodilators include, for example, hydralazine,nitroglycerin, and isosorbide dinitrate.

[0102] Suitable diuretics include, for example, furosemide, diuril,amiloride, and hydrodiuril.

[0103] Suitable α-adrenergic receptor antagonists include, for example,prazosin, doxazocin, and labetalol.

[0104] Suitable antioxidants include vitamin E, vitamin C, andisoflavones.

[0105] A compound suitable for use in methods of the invention and atherapeutic cardiovascular compound are administered concurrently.“Concurrent administration” and “concurrently administering” as usedherein includes administering a compound suitable for use in methods ofthe invention and a therapeutic cardiovascular compound in admixture,such as, for example, in a pharmaceutical composition or in solution, oras separate compounds, such as, for example, separate pharmaceuticalcompositions or solutions administered consecutively, simultaneously, orat different times but not so distant in time such that the compoundsuitable for use in methods of the invention and the therapeuticcardiovascular compound cannot interact and a lower dosage amount of theactive ingredient cannot be administered.

[0106] A physician or veterinarian of ordinary skill readily determinesa subject who is exhibiting symptoms of any one or more of the diseasesdescribed above. Regardless of the route of administration selected, thecompound suitable for use in methods of the invention and thetherapeutic cardiovascular compound are formulated into pharmaceuticallyacceptable unit dosage forms by conventional methods known to thepharmaceutical art. An effective but nontoxic quantity of the compoundsuitable for use in methods of the invention and the therapeuticcardiovascular compound are employed in the treatment.

[0107] The compound suitable for use in methods of the invention and thetherapeutic cardiovascular compound may be concurrently administeredenterally and/or parenterally in admixture or separately. Parenteraladministration includes subcutaneous, intramuscular, intradermal,intramammary, intravenous, and other administrative methods known in theart. Enteral administration includes tablets, sustained release tablets,enteric coated tablets, capsules, sustained release capsules, entericcoated capsules, pills, powders, granules, solutions, and the like.

[0108] A pharmaceutical composition suitable for administrationcomprises a pharmaceutically acceptable carrier and a compound suitablefor use in methods of the invention and/or a therapeutic cardiovascularcompound. The pharmaceutical composition comprises a pharmaceuticallyacceptable carrier and a compound suitable for use in methods of theinvention, such as, for example, pyridoxal-5′-phosphate, pyridoxal,pyridoxamine, a 3-acylated pyridoxal analogue, a pharmaceuticallyacceptable acid addition salt thereof, and a mixture thereof. Apharmaceutically acceptable carrier includes, but is not limited to,physiological saline, ringers, phosphate buffered saline, and othercarriers known in the art. Pharmaceutical compositions may also includestabilizers, antioxidants, colorants, and diluents. Pharmaceuticallyacceptable carriers and additives are chosen such that side effects fromthe pharmaceutical compound are reduced or minimized and the performanceof the compound is not canceled or inhibited to such an extent thattreatment is ineffective.

[0109] Methods of preparing pharmaceutical compositions containing apharmaceutically acceptable carrier and a compound suitable for use inmethods of the invention and/or a therapeutic cardiovascular compoundare known to those of skill in the art. All methods may include the stepof bringing the compound suitable for use in methods of the inventionand/or the therapeutic cardiovascular compound in association with thecarrier or additives. In general, the formulations are prepared byuniformly and intimately bringing the compound into association with aliquid carrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired unit dosage form.

[0110] The ordinarily skilled physician or veterinarian will readilydetermine and prescribe the therapeutically effective amount of thecompound to treat the disease for which treatment is administered. In soproceeding, the physician or veterinarian could employ relatively lowdosages at first, subsequently increasing the dose until a maximumresponse is obtained. Typically, the particular disease, the severity ofthe disease, the compound to be administered, the route ofadministration, and the characteristics of the mammal to be treated, forexample, age, sex, and weight, are considered in determining theeffective amount to administer. Administering a therapeuticallyeffective amount of a compound suitable for use in methods of theinvention to treat cardiovascular and related diseases as well assymptoms thereof is typically in a range of about 0.1-100 mg/kg of apatient's body weight, more preferably in the range of about 0.5-50mg/kg of a patient's body weight per daily dose when administered alone.The compound suitable for use in methods of the invention may beadministered for periods of short and long duration.

[0111] Therapeutically effective amounts of respective therapeuticcardiovascular compounds when administered as sole active ingredientsare known in the art and may be found in, for example, Physicians' DeskReference (53^(rd) ed., 1999).

[0112] When concurrently administering a compound suitable for use inmethods of the invention and a therapeutic cardiovascular compound, thecompound suitable for use in methods of the invention is typicallyadministered in a range of about 0.1-100 mg/kg of a patient's bodyweight, preferably 0.5-50 mg/kg of a patient's body weight, per dailydose, and the therapeutic cardiovascular compound is administered in anamount less than the amount known in the art, which is administered whenthe therapeutic cardiovascular compound is administered as the soleactive ingredient. Typically, the therapeutic cardiovascular compound isadministered in an amount at least 5% less than the amount known in theart, which is administered when the therapeutic cardiovascular compoundis administered as the sole active ingredient.

[0113] A therapeutically effective amount of a compound suitable for usein methods of the invention and a therapeutic cardiovascular compoundfor treating cardiovascular and related diseases and symptoms thereofcan be administered prior to, concurrently with, or after the onset ofthe disease or symptom. For example, a therapeutically effective amountof a compound suitable for use in methods of the invention and atherapeutic cardiovascular compound for treating ischemia reperfusioninjury or myocardial infarction can be administered before, during, orfollowing ischemia (including during or following reperfusion), as wellas continually for some period spanning from pre- to post-ischemia. Forexample, the compound suitable for use in methods of the invention and atherapeutic cardiovascular compound may be concurrently administeredprior to heart procedures, including bypass surgery, thrombolysis, andangioplasty, and prior to any other procedures that require blood flowbe interrupted and then resumed. Additionally, a compound suitable foruse in methods of the invention and a therapeutic cardiovascularcompound may be taken on a regular basis to protect against cellulardysfunction arising from arrhythmia and heart failure.

[0114] The invention is further elaborated by the representativeexamples as follows. Such examples are not meant to be limiting.

EXAMPLES Example 1 Synthesis of morpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine)

[0115] A mixture of morpholine (20 g) and toluene (100 mL) was stirredand heated using an oil bath set to 100° C. for 15 minutes. Pyridoxalhydrochloride (10 g) was then added and the reaction mixture was stirredat 100° C. for two hours. The reaction mixture was then concentrated bydistillation of the toluene and morpholine. The concentrated reactionmixture was washed three times by adding toluene (100 mL) and removingthe toluene by distillation. After washing, the residue was dissolved intoluene and filtered, and then hexane was added until precipitationbegan, at which time the reaction mixture was left overnight at roomtemperature. Crystals were collected and washed thoroughly with hexane.

[0116] Nuclear magnetic resonance spectroscopy (NMR) and massspectroscopy confirmed the identity of the synthesized compound. Thepurity of the compound was analyzed by high performance liquidchromatography (HPLC) using a C-18 reverse phase column andwater/acetonitrile as solvent (1-100% acetonitrile over 25 minutes).

Example 2 Synthesis of the 3-toluate of the morpholinepyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-(p-toluoyloxy)-6-methylfuro(3,4-c)pyridine)

[0117] Anhydrous powdered potassium carbonate (5 g), acetone (100 mL),and morpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine) (1.11g, 5 mmoles) were mixed in a nitrogen-cooled, dry flask. The reactionmixture was cooled to between 0 and 5° C. and then p-toluoyl chloride(1.06 g, 6 mmoles) in acetone (20 mL) was added. This mixture wasstirred for two hours, followed by filtering out the solid andevaporating the solution to dryness under vacuum. The residue waschromatographed on silica gel using a mixture of ethyl acetate andhexane as solvent.

[0118] The purified solid was analyzed by thin layer chromatography(TLC), NMR, and mass spectroscopy. The purity of the synthesizedcompound was confirmed by HPLC as described in Example 1.

Example 3 Synthesis of the 3-toluate of pyridoxal(2-methyl-3-toluoyloxy-4-formyl-5-hydroxymethylpyridine)

[0119] Anhydrous potassium carbonate (10 g), acetone (100 mL), andpyridoxal hydrochloride (2.03 g, 10 mmoles) were mixed in anitrogen-cooled, dry flask. The mixture was cooled to between 0 and 5°C. and then p-toluoyl chloride (2.12 g, 12 mmoles) in acetone (20 mL)was added. The reaction mixture was stirred for two hours followed byfiltering out the solid and evaporating the solution to dryness undervacuum. The residue was chromatographed on silica gel as described inExample 2.

[0120] The purified solid was analyzed by TLC, NMR, and massspectroscopy. The purity of the compound was confirmed by HPLC asdescribed in Example 1.

[0121] Alternative to the above-described method, the 3-toluate ofpyridoxal is synthesized by reacting the compound of Example 2 with 80%aqueous acetic acid at 60° C. for 30 minutes, and then diluting withwater and extracting by ethyl acetate. The ethyl acetate layer is washedwith 5% aqueous sodium bicarbonate, dried with magnesium sulfate, andevaporated to dryness. The compound is also analyzed as described supra.

Example 4 Synthesis of 3-β-naphthoate of the morpholinepyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-(β-naphthoyloxy)-6-methylfuro(3,4-c)pyridine)

[0122] Anhydrous powdered potassium carbonate (5 g), acetone (100 mL),and morpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine) (1.11g, 5 mmoles) were mixed in a nitrogen-cooled, dry flask. The mixture wascooled to between 0 and 5° C. and then β-naphthoyl chloride (1.06 g, 6mmoles) in acetone (20 mL) was added. The reaction mixture was stirredfor two hours, and then the solid was filtered out and the solution wasevaporated to dryness under vacuum. The residue was chromatographedaccording to Example 2.

[0123] The purified solid was analyzed according to Example 2, and thepurity was confirmed according to Example 1.

Example 5 Synthesis of the 3-β-naphthoate of pyridoxal(2-methyl-3-β-naphthoyloxy-4-formyl-5-hydroxymethylpyridine)

[0124] Anhydrous potassium carbonate (10 g), acetone (100 mL), andpyridoxal hydrochloride (2.03 g, 10 mmoles) were mixed in anitrogen-cooled, dry flask. The mixture was cooled to between 0 and 5°C. and then β-naphthoyl chloride (2.12 g, 12 mmoles) in acetone (20 mL)was added and the mixture was stirred for two hours. The solid wasfiltered out and the solution was evaporated to dryness under vacuum.The residue was chromatographed according to Example 2.

[0125] The purified solid was analyzed according to Example 2, and thepurity was confirmed according to Example 1.

[0126] Alternative to the above-described synthesis, the 3-β-naphthoateof pyridoxal is prepared by reacting the compound of Example 4 with 80%aqueous acetic acid at 60° C. for 30 minutes, followed by diluting withwater and extracting by ethyl acetate. The ethyl acetate layer is thenwashed with 5% aqueous sodium bicarbonate, dried with magnesium sulfate,and evaporated to dryness. The compound is also analyzed as describedsupra.

Example 6 Synthesis of 3-pivaloyl of the morpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-pivaloyloxy)-6-methylfuro (3,4-c)pyridine)

[0127] Anhydrous powdered potassium carbonate (5 g), acetone (100 mL),and morpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine) (1.11μg, 5 mmoles) were mixed in a nitrogen-cooled, dry flask. The mixturewas cooled to between 0 and 5° C. and then pivaloyl chloride(trimethylacetyl chloride) (720 mg, 6 mmoles) in acetone (20 mL) wasadded. The reaction mixture was stirred for two hours. The solid wasthen filtered out and the solution was evaporated to dryness undervacuum. The residue was chromatographed according to Example 2.

[0128] The purified solid was analyzed according to Example 2, and thepurity was confirmed according to Example 1.

Example 7 Synthesis of 3-dimethylcarbamoyl of the morpholinepyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-(dimethylcarbamoyloxy)-6-methylfuro(3,4-c)pyridine)

[0129] Anhydrous powdered potassium carbonate (5 g), acetone (100 mL),and morpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine) (1.11g, 5 mmoles) were mixed in a nitrogen-cooled, dry flask. The mixture wascooled to between 0 and 5° C. and then dimethylcarbamoyl chloride (642mg, 6 mmoles) in acetone (20 mL) was added. The reaction mixture wasstirred for two hours. The solid was then filtered out and the solutionwas evaporated to dryness under vacuum. The residue was chromatographedaccording to Example 2.

[0130] The purified solid was analyzed according to Example 2, and thepurity was confirmed according to Example 1.

Example 8 Synthesis of 3-acetylsalicyloyl of the morpholinepyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-acetylsalicyloxy)-6-methylfuro(3.4-c)pyridine)

[0131] Anhydrous powdered potassium carbonate (5 g), acetone (100 mL),and morpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine) (1.11g, 5 mmoles) were mixed in a nitrogen-cooled, dry flask. The mixture wascooled to between 0 and 5° C. and then acetylsalicyloyl chloride (1.09g, 6 mmoles) in acetone (20 mL) was added. The reaction mixture wasstirred for two hours. The solid was then filtered out and the solutionwas evaporated to dryness under vacuum. The residue was chromatographedaccording to Example 2.

[0132] The purified solid was analyzed according to Example 2, and thepurity was confirmed according to Example 1.

Example 9 In Vitro—Ischemia Reperfusion in Isolated Rat Hearts andMeasurement of Left Ventricular Developed Pressure (LVDP)

[0133] Male Sprague-Dawley rats weighing 250-300 g are anaesthetizedwith a mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg). Thehearts are rapidly excised, cannulated to a Langendorff apparatus andperfused with Krebs-Henseleit-solution, gassed with a mixture of 95% O2and 5% CO2, pH 7.4. The perfusate contained (in mM): 120 NaCl, 25NaHCO3, 11 glucose, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4 and 1.25 CaCl2.

[0134] The hearts are electrically stimulated at a rate of 300 beats/min(Phipps and Bird Inc., Richmond, Va.) and a water-filled latex balloonis inserted in the left ventricle and connected to a pressure transducer(Model 1050BP; BYOPAC SYSTEM INC., Goleta, Calif.) for the leftventricular systolic measurements. The left ventricular end diastolicpressure (LVEDP) is adjusted at 10 mmHg at the beginning of theexperiment. In some experiments the left ventricular pressures aredifferentiated to estimate the rate of ventricular contraction (+dP/dt)and rate of ventricular relaxation (−dP/dt) using the Acknowledge 3.03software for Windows (BIOPAC SYSTEM INC.,) Goleta, Calif.). All heartsare stabilized for a period of 30 min and then randomly distributed intonine different experimental groups (n=5-8 per group). The experimentalgroups are defined as follows:

[0135] 1) Control group (control hearts are further perfused for 90minutes for a total of 130 min of continous perfusion);

[0136] 2) Ischemia reperfusion group (Ischemia reperfusion hearts aremade globally ischemic by stopping the coronary flow completely for 30min and then the hearts are reperfused for 60 min);

[0137] 3) P-5-P (15 uM) treated group;

[0138] 4) captopril (100 uM) treated group;

[0139] 5) verapamil (0.01 uM)) treated group;

[0140] 6) propranolol (3 mM) treated group;

[0141] 7) PPADS (10 uM) treated group;

[0142] 8) P-5-P+captopril treated group;

[0143] 9) P-5-P+verapamil treated group;

[0144] 10) P-5-P+propranolol treated group;

[0145] 11) P-5-P+PPADS treated group.

[0146] Drug treatment is started 10 min before global ischemia followedby 30 min global ischemia and 60 min reperfusion. At the end of someexperiments, the hearts are quickly freeze-clamped with a liquidnitrogen precooled Wollenberger tong. Rats are housed in clear cages ina temperature and humidity controlled room on a 12 hr light-dark cycle.Food and water are supplied ad libitum.

[0147] Hearts subject to 30 min of ischemia followed by 60 min ofreperfusion showed slight recovery in the contractile function asrepresented by 29.5% recovery in LVDP (left ventricular developedpressure). As compared to the untreated group, treatment with P-5-P,captopril, or P-5-P and captopril showed better recoveries in LVDP by78.2%, 61.4%, and 132% respectively (Table I). TABLE I Effect ofPyridoxal-5-phosphate (P-5-P, 15 uM) and Captopril (100 uM) on %recovery of left ventricular systolic pressure (LVDP). LVDP LVEDP LVSP %recovery Drugs (B) (A) mmHg mmHg (LVDP) Unreated 87 ± 7   25 ± 2.9 62 ±5.6 87 ± 6.9 29.5 ± 3.7 P5P  80 ± 3.8 63 ± 5  35 ± 4.8 98 ± 8.2  78.2 ±3.3* Captopril  78 ± 10.9  47 ± 8.6 54 ± 6.7 101 ± 14.6  61.4 ± 5.2*P5P +  89 ± 6.9  69 ± 7.4 28 ± 7.3 117 ± 8.4   ¹² 132 ± 7.5^(#)Captopril

[0148] Hearts subject to 30 min of ischemia followed by 60 min ofreperfusion showed slight recovery in the contractile function asrepresented by 29.5% recovery in LVDP. As compared to the untreatedgroup, treatment with P-5-P, verapamil, or P-5-P and verapamil showedbetter recoveries in LVDP by 78.2%, 43%, and 109% respectively (TableII). TABLE II Effect of Pyridoxal-5-phosphate (P-5-P,l5uM) and Verapamil(0.01 uM) on % recovery of left ventricular systolic pressure (LVDP).LVDP LVEDP LVSP % recovery Drugs (B) (A) mmHg mmHg (LVDP) Untreated 87 ±7   25 ± 2.9 62 ± 5.6 87 ± 6.9 29.5 ± 3.7  P5P  80 ± 3.8 63 ± 5  35 ±4.8 98 ± 8.2 78.2 ± 3.3* Verapamil  54 ± 9.1  23 ± 4.5 55 ± 5.1 78 ± 7.7 43 ± 6.6 P5P +   78 ± 10.5   85 ± 11.7 34 ± 7.3 119 ± 8   109 ± 4.6^(#)Verapamil

[0149] Hearts subject to 30 min of ischemia followed by 60 min ofreperfusion showed slight recovery in the contractile function asrepresented by 29.5% recovery in LVDP. As compared to the untreatedgroup, treatment with P-5-P, PPADS, or P-5-P and PPADS showed betterrecoveries in LVDP by 78.2%, 61%, and 128% respectively (Table III).TABLE III Effect of Pyridoxal-5-phosphate (P-5-P, 15 uM) and Pyridoxalphosphate 6-azophenyl-2'-4'disulfonic acid (PPADS 100 uM) on % recoveryof left ventricular systolic pressure (LVDP). LVDP LVEDP LVSP % recoveryDrugs (B) (A) mmHg mmHg (LVDP) Untreated 87 ± 7   25 ± 2.9 62 ± 5.6  87± 6.9 29.5 ± 3.7  P5P  80 ± 3.8 63 ± 5  35 ± 4.8  98 ± 8.2 78.2 ± 3.3PPADS   92 ± 15.2   58 ± 13.6 57 ± 6.3  115 ± 11.5   61 ± 4.8* P5P +  82 ± 15.8  105 ± 22.8 34 ± 3.1  139 ± 21.6   128 ± 13.8# PPADS

[0150] Hearts subject to 30 min of ischemia followed by 60 min ofreperfusion showed slight recovery in the contractile function asrepresented by 29.5% recovery in LVDP. As compared to the untreatedgroup, treatment with P-5-P, propranolol, or P-5-P and propranololshowed better recoveries in LVDP by 78.2%, 74%, and 120% respectively(Table IV). TABLE IV Effect of Pyridoxal-5-phosphate (P-5-P, 15 uM) andPropranolol (3 uM) on % recovery of left ventricular systolic pressure(LVDP). LVDP LVEDP LVSP % recovery Drugs (B) (A) mmHg mmHg (LVDP)Untreated 87 ± 7   25 ± 2.9 62 ± 5.6  87 ± 6.9 29.5 ± 3.7  P5P  80 ± 3.863 ± 5  35 ± 4.8  98 ± 8.2 78.2 ± 3.3* Propranolol   61 ± 10.8  45 ± 9.727 ± 6.6   72 ± 15.1   74 ± 4.9* P5P +   67 ± 12.6   75 ± 10.4 40 ± 4.2115 ± 8.3    120 ± 15.5^(#) Propranolol

[0151] Tables I-IV demonstrate that P-5-P in addition to providingsignificant benefit in ischemia reperfusion injury when given alone alsoimproves or adds to the benefits associated with other commonly useddrugs when given in combination with these drugs.

[0152] In addition to captopril, other angiotensin converting enzymeinhibitors, such as, for example, enalapril or imidapril, can similarlybe administered in place of captopril. In addition to verapamil, otherknown calcium channel blockers, such as, for example, nifedipine ordiltiazem, can similarly be administered in place of verapamil. Inaddition to propranolol, other β-adrenergic receptor antagonists suchas, for example, atenolol, timolol, and metoprolol can similarly beadministered in place of propranolol. Additionally, angiotensin IIreceptor antagonists such as, for example, losartan and valsartan can beused in the above example.

Example 10 In Vivo—Coronary Artery Ligation

[0153] Myocardial infarction is produced in male Sprague-Dawley rats(200-250 g) by occlusion of the left coronary artery as described inSethi et al., J. Cardiac Failure, 1(5) (1995) and Sethi et al., Am. J.Physiol., 272 (1997).

[0154] Rats are anesthetized with 1-5% isoflurane in 100% 0₂ (2L flowrate). The skin is incised along the left sterna border and the 4th ribis cut proximal to the sternum and a retractor inserted. The pericardialsac is opened and the heart externalized. The left anterior descendingcoronary artery is ligated approximately 2 mm from its origin on theaorta using a 6-0 silk suture. The heart is then repositioned in thechest and the incision closed via purse-string sutures.

[0155] Sham operated rats undergo identical treatment except that theartery is not ligated. Mortality due to surgery is less than 1%. Unlessindicated in the text, the experimental animals showing infarctsize >30% of the left ventricle are used in this study. All animals areallowed to recover, allowed to receive food and water ad libitum, andare maintained for a period of 21 days for Electrocardiogram (ECG),hemodynamic, and histological assessment.

[0156] Occlusion of the coronary artery in rats has been shown toproduce myocardial cell damage which results in scar formation in theleft ventricle and heart dysfunction. While the complete healing of thescar occurs within 3 weeks of the coronary occlusion, mild, moderate andsevere stages of congestive heart failure have been reported to occur at4, 8 and 16 weeks after ligation. Accordingly, the contractiledysfunction seen at 3 weeks after the coronary occlusion in rats is dueto acute ischemic changes.

[0157] The rats are housed in clear cages in a temperature and humiditycontrolled room, on a 12 hour light-dark cycle. Food and water aresupplied ad libitum. After surgery, rats are randomly assigned totreatment or non-treatment in both sham and experimental groups.Randomization of animals was performed and treatment begins 1 hour aftercoronary occlusion and continues for 21 days. The total duration ofexperiments in each case is 21 days. The groups are as follows:

[0158] 1) sham operated;

[0159] 2) coronary artery ligated (treatment with equal volumes ofsaline);

[0160] 3) coronary artery ligated (treated with 10 mg/kg P-5-P);

[0161] 4) coronary artery ligated (treated with 100 mg/kg captopril);

[0162] 5) coronary artery ligated (treated with 50 mg/kg propranolol);

[0163] 6) coronary artery ligated (treated with 100 mg/kg aspirin);

[0164] 7) coronary artery ligated (treated with 25 mg/kg verapamil);

[0165] 8) coronary artery ligated (treated with 10 mg/kg P-5-P+100 mg/kgcaptopril);

[0166] 9) coronary artery ligated (treated with 10 mg/kg P-5-P+50 mg/kgpropranolol);

[0167] 10) coronary artery ligated (treated with 10 mg/kg P-5-P+100mg/kg aspirin);

[0168] 11) coronary artery ligated (treated with 10 mg/kg P-5-P+25 mg/kgverapamil).

[0169] P-5-P (10 mg/kg), captopril (100 mg/kg), propranolol (50 mg/kg),verapamil (25 mg/kg) and aspirin (100 mg/kg) were administered oncedaily by gastric tube.

[0170] Acute myocardial infarction resulted in a total mortality of 35%% in the untreated group of rats in 21 days. The highest mortalityoccurred within the first 2 days following occlusion. As compared to theuntreated group, treatment with P-5-P, aspirin, or P-5-P and aspirinshowed lower mortality rates of 15%, 25%, 15%, respectively (FIG. 1).

[0171] Acute myocardial infarction resulted in a total mortality of 35%% in the untreated group of rats in 21 days. The highest mortalityoccurred within the first 2 days following occlusion. As compared to theuntreated group, treatment with P-5-P, captopril, or P-5-P and captoprilshowed lower mortality rates of 10%, 15%, 20%, respectively (FIG. 2).

[0172] Acute myocardial infarction resulted in a total mortality of 35%% in the untreated group of rats in 21 days. The highest mortalityoccurred within the first 2 days following occlusion. As compared to theuntreated group, treatment with P-5-P, propranolol, or P-5-P andpropranolol showed lower mortality rates of 15%, 20%, 20%, respectively(FIG. 3).

[0173] Acute myocardial infarction resulted in a total mortality of 35%% in the untreated group of rats in 21 days. The highest mortalityoccurred within the first 2 days following occlusion. As compared to theuntreated group, treatment with P-5-P, verapamil, or P-5-P and verapamilshowed lower mortality rates of 15%, 25%, 10%, respectively (FIG. 4).

[0174] In addition to captopril, other angiotensin converting enzymeinhibitors, such as, for example, enalapril or imidapril, can similarlybe administered in place of captopril. In addition to verapamil, otherknown calcium channel blockers, such as, for example, nifedipine ordiltiazem, can similarly be administered in place of verapamil. Inaddition to propranolol, other β-adrenergic receptor antagonists suchas, for example, atenolol, timolol, and metoprolol can similarly beadministered in place of propranolol. In addition to aspirin, otherantithrombolytic agents such as, for example, antiplatelet agents andheparin can similarly be administered in place of aspirin. Additionally,angiotensin II receptor antagonists such as, for example, losartan andvalsartan can be used in the above example.

[0175] These animals are used in Examples 11 and 12 below. For EKGstudies, these animals are used as their controls before surgery, sothat before surgery is done on these animals EKG traces are taken whichare then used as controls for the same animals after surgery.

Example 11 In Vivo—Hemodynamic Changes

[0176] The animals are prepared and grouped as described in Example 10and were anesthetized with a solution of ketamine/xylazine which wasinjected. To maintain adequate ventilation, the trachea was intubated;the right carotid artery was exposed for introducing a microtip pressuretransducer (model SPR-249, Millar, Houston, Tex.) into the leftventricle. The catheter was secured with a silk ligature around theartery, and various hemodynamic parameters such as left ventricularsystolic pressure (LVSP), left ventricular end diastolic pressure(LVEDP), rate of contraction (+dp/dt), rate of relaxation (−dP/dt) wererecorded and calculated on a computer system using a Acknowledge 3.1software.

[0177] Once the hemodynamic parameters were measured the animals weresacrificed and hearts removed for measurement of heart weight, rightventricular weight, left ventricular weight and scar weight. Becausecomplete healing of the scar in rats after coronary occlusion requiresapproximately 3 weeks, scar weight were measured only at 21 days.

[0178] FIGS. 5-8 demonstrate that the occlusion of coronary artery inrats for 21 days produces a significant scar evident by scar weight.Furthermore, FIGS. 5-8 demonstrate that P-5-P has a significantbeneficial effect on scar weight in groups where P-5-P treatment iseither given alone or in combination with verapamil, aspirin, captopril,or propranolol, respectively.

[0179] FIGS. 9-12 demonstrate that P-5-P has a significant beneficialeffect on rate of contraction (+dP/dt) in groups where P-5-P treatmentis either given alone or in combination with verapamil, aspirin,captopril, or propranolol, respectively.

[0180] FIGS. 13-16 demonstrate that P-5-P has a significant beneficialeffect on rate of relaxzation (+dP/dt) in groups where P-5-P treatmentis either given alone or in combination with verapamil, aspirin,captopril, or propranolol, respectively.

[0181] FIGS. 17-20 demonstrate that P-5-P has a significant beneficialeffect on rate of left ventricular end diastolic pressure (LVEDP) ingroups where P-5-P treatment is either given alone or in combinationwith verapamil, aspirin, captopril, or propranolol, respectively.

[0182] FIGS. 21-24 demonstrate that P-5-P has a significant beneficialeffect on whole heart weight in groups where P-5-P treatment is eithergiven alone or in combination with verapamil, aspirin, captopril, orpropranolol, respectively.

[0183] FIGS. 25-28 demonstrate that P-5-P has a significant beneficialeffect on right ventricular weight in groups where P-5-P treatment iseither given alone or in combination with verapamil, aspirin, captopril,or propranolol, respectively.

[0184] In addition to captopril, other angiotensin converting enzymeinhibitors, such as, for example, enalapril or imidapril, can similarlybe administered in place of captopril. In addition to verapamil, otherknown calcium channel blockers, such as, for example, nifedipine ordiltiazem, can similarly be administered in place of verapamil. Inaddition to propranolol, other β-adrenergic receptor antagonists suchas, for example, atenolol, timolol, and metoprolol can similarly beadministered in place of propranolol. In addition to aspirin, otherantithrombolytic agents such as, for example, antiplatelet agents andheparin can similarly be administered in place of aspirin. Additionally,angiotensin II receptor antagonists such as, for example, losartan andvalsartan can be used in the above example.

Example 12 In Vivo—Hypertension

[0185] It has been well demonstrated by various investigators thatfeeding 8-10% sucrose in water induces hypertension in rats. Zein etal., Am. Coll. Nutr., 17 (1), 36-37, 1998; Hulman et al., Pediatr. Res.,36:95-101; Reaven et al., Am. J. Hypertens; 1991:610-614. In applyingthis model, the concurrent administration of pyridoxal-5′-phosphate andcaptopril or verapamil significantly decreases the sucrose-inducedincrease in systolic blood pressure (SBP).

[0186] The blood pressure is monitored using the tail cuff method. TheSBP is detected on an amplifier and the Acknowledge™ computer softwareprogram is used to determine the calculations.

[0187] The effect of concurrent administration of pyridoxal-5′-phosphateand captopril or verapamil on systolic blood pressure (marker ofhypertension) in 10% sucrose induced hypertension in rats is determined.

[0188]FIGS. 29A and 9B demonstrate that P-5-P has a significantbeneficial effect on systolic blood pressure in groups where P-5-Ptreatment is either given alone or in combination with verapamil orcaptopril 1 week prior to inducing hypertension in rats with a sucrosediet.

[0189]FIGS. 29A and 29B demonstrate that P-5-P has a significantbeneficial effect on systolic blood pressure in groups where P-5-Ptreatment is either given alone or in combination with verapamil orcaptopril 1 week prior to inducing hypertension in rats with a sucrosediet.

[0190]FIGS. 30A and 30B demonstrate that P-5-P has a significantbeneficial effect on systolic blood pressure in groups where P-5-Ptreatment is either given alone or in combination with verapamil orcaptopril the same day as inducing hypertension in rats with a sucrosediet.

[0191]FIGS. 31A and 31B demonstrate that P-5-P has a significantbeneficial effect on systolic blood pressure in groups where P-5-Ptreatment is either given alone or in combination with verapamil orcaptopril two weeks after inducing hypertension in rats with a sucrosediet.

[0192] In addition to captopril, other angiotensin converting enzymeinhibitors, such as, for example, enalapril or imidapril, can similarlybe administered in place of captopril. In addition to verapamil, otherknown calcium channel blockers, such as, for example, nifedipine ordiltiazem, can similarly be administered in place of verapamil. Inaddition to propranolol, other β-adrenergic receptor antagonists suchas, for example, atenolol, timolol, and metoprolol can similarly beadministered in place of propranolol. Additionally, angiotensin IIreceptor antagonists such as, for example, losartan and valsartan can beused in the above example.

[0193] It should be noted that, as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to a composition containing “a compound” includes amixture of two or more compounds.

[0194] Although embodiments of the invention have been described above,it is not limited thereto, and it will be apparent to persons skilled inthe art that numerous modifications and variations form part of thepresent invention insofar as they do not depart from the spirit, nature,and scope of the claimed and described invention.

[0195] All references, applications, and patents cited herein areincorporated by reference.

We claim:
 1. A method of treating ischemia in a mammal comprising:concurrently administering to the mammal a therapeutically effectiveamount of a combination of a compound selected from the group consistingof pyridoxal-5′-phosphate, pyridoxal, pyridoxamine, a 3-acylatedpyridoxal analogue, a pharmaceutically acceptable acid addition saltthereof, and a mixture thereof, and a therapeutic cardiovascularcompound selected from the group consisting of an angiotensin convertingenzyme inhibitor, an angiotensin II receptor antagonist, a calciumchannel blocker, an antithrombolytic agent, a β-adrenergic receptorantagonist, a diuretic, an α-adrenergic receptor antagonist, and amixture thereof.
 2. A method according to claim 1, wherein the3-acylated pyridoxal analogue is a compound of the formula

wherein R₁ is a straight or branched alkyl group, a straight or branchedalkenyl group, in which an alkyl or alkenyl group may be interrupted bya nitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or anunsubstituted or substituted aryl group.
 3. A method according to claim1, wherein the 3-acylated pyridoxal analogue is a compound of theformula

wherein R₁ is a straight or branched alkyl group, a straight or branchedalkenyl group, in which an alkyl or alkenyl group may be interrupted bya nitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or anunsubstituted or substituted aryl group; and R₂ is a secondary aminogroup.
 4. A method according to claim 1, wherein the angiotensinconverting enzyme inhibitor is captopril, enalapril, lisinopril,benazapril, fosinopril, quinapril, ramipril, spirapril, imidapril, ormoexipril.
 5. A method according to claim 1, wherein the angiotensin IIreceptor antagonist is losartan or valsartan.
 6. A method according toclaim 1, wherein the calcium channel blocker is verapamil, diltiazem,nicardipine, nifedipine, amlodipine, felodipine, nimodipine, orbepridil.
 7. A method according to claim 1, wherein the antithrombolyticagent is an antiplatelet agent, aspirin, or heparin.
 8. A methodaccording to claim 1, wherein the β-adrenergic receptor antagonist isatenolol, propranolol, timolol, or metoprolol.
 9. A method according toclaim 1, wherein the diuretic is furosemide, diuril, amiloride, orhydrodiuril.
 10. A method according to claim 1, wherein the compound isadministered enterally or parenterally and the therapeuticcardiovascular compound is administered enterally or parenterally.
 11. Amethod according to claim 1, wherein the compound and the therapeuticcardiovascular compound are administered in a single dosage form.
 12. Amethod of treating ischemia reperfusion injury in a mammal comprising:concurrently administering to the mammal a therapeutically effectiveamount of a combination of a compound selected from the group consistingof pyridoxal-5′-phosphate, pyridoxal, pyridoxamine, a 3-acylatedpyridoxal analogue, a pharmaceutically acceptable acid addition saltthereof, and a mixture thereof, and a therapeutic cardiovascularcompound selected from the group consisting of an angiotensin convertingenzyme inhibitor, an angiotensin II receptor antagonist, a calciumchannel blocker, and a mixture thereof.
 13. A method according to claim12, wherein the 3-acylated pyridoxal analogue is a compound of theformula

wherein R₁ is a straight or branched alkyl group, a straight or branchedalkenyl group, in which an alkyl or alkenyl group may be interrupted bya nitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or anunsubstituted or substituted aryl group.
 14. A method according to claim12, wherein the 3-acylated pyridoxal analogue is a compound of theformula

wherein R₁ is a straight or branched alkyl group, a straight or branchedalkenyl group, in which an alkyl or alkenyl group may be interrupted bya nitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or anunsubstituted or substituted aryl group; and R₂ is a secondary aminogroup.
 15. A method according to claim 12, wherein the angiotensinconverting enzyme inhibitor is captopril, enalapril, lisinopril,benzazpril, fosinopril, quinapril, ramipril, spirapril, imidapril, ormoexipril.
 16. A method according to claim 12, wherein the angiotensinII receptor antagonist is losartan or valsartan.
 17. A method accordingto claim 12, wherein the calcium channel blocker is verapamil,diltiazem, nicardipine, nifedipine, amlodipine, felodipine, nimodipine,or bepridil.
 18. A method according to claim 12, wherein the compound isadministered enterally or parenterally and the therapeuticcardiovascular compound is administered enterally or parenterally.
 19. Amethod according to claim 12, wherein the compound and the therapeuticcardiovascular compound are administered in a single dosage form.
 20. Amethod of treating myocardial ischemia in a mammal comprising:concurrently administering to the mammal a therapeutically effectiveamount of a combination of a compound selected from the group consistingof pyridoxal-5′-phosphate, pyridoxal, pyridoxamine, a 3-acylatedpyridoxal analogue, a pharmaceutically acceptable acid addition saltthereof, and a mixture thereof, and a therapeutic cardiovascularcompound selected from the group consisting of an angiotensin convertingenzyme inhibitor, an angiotensin II receptor antagonist, a calciumchannel blocker, an antithrombolytic agent, a β-adrenergic receptorantagonist, a diuretic, an α-adrenergic receptor antagonist, and amixture thereof.
 21. A method according to claim 20, wherein the3-acylated pyridoxal analogue is a compound of the formula

wherein R₁ is a straight or branched alkyl group, a straight or branchedalkenyl group, in which an alkyl or alkenyl group may be interrupted bya nitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or anunsubstituted or substituted aryl group.
 22. A method according to claim20, wherein the 3-acylated pyridoxal analogue is a compound of theformula

wherein R₁ is a straight or branched alkyl group, a straight or branchedalkenyl group, in which an alkyl or alkenyl group may be interrupted bya nitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or anunsubstituted or substituted aryl group; and R₂ is a secondary aminogroup.
 23. A method according to claim 20, wherein the angiotensinconverting enzyme inhibitor is captopril, enalapril, lisinopril,benazapril, fosinopril, quinapril, ramipril, spirapril, imidapril, ormoexipril.
 24. A method according to claim 20, wherein the angiotensinII receptor antagonist is losartan or valsartan.
 25. A method accordingto claim 20, wherein the calcium channel blocker is verapamil,diltiazem, nicardipine, nifedipine, amlodipine, felodipine, nimodipine,or bepridil.
 26. A method according to claim 20, wherein theantithrombolytic agent is an antiplatelet agent, aspirin, or heparin.27. A method according to claim 20, wherein the β-adrenergic receptorantagonist is atenolol, propranolol, timolol, or metoprolol.
 28. Amethod according to claim 20, wherein the diuretic is furosemide,diuril, amiloride, or hydrodiuril.
 29. A method according to claim 20,wherein the compound is administered enterally or parenterally and thetherapeutic cardiovascular compound is administered enterally orparenterally.
 30. A method according to claim 20, wherein the compoundand the therapeutic cardiovascular compound are administered in a singledosage form.